It has become increasingly clear that the same enzymes that participate in blood coagulation and fibrinolysis also mediate additional and disparate biologic functions. With surprising analogies to the mechanisms of hormone-mediated growth factor activity, thrombin exerts a potent mitogenic effect on various cell types in a reaction exquisitely coordinated by specific cellular receptors (Chen, L. B., et al., Proc. Natl. Acad. Sci. USA, 72: 131 (1975); Glenn, K. C., et al., Nature, 278: 711 (1979); Baker, J. B., et al., Nature, 278: 743 (1979)). Similarly, the delicate balance between mitogenesis, malignant transformation, protooncogene expression, and cell differentiation has been shown to be profoundly influenced by protease activity (Unkeless, J. C., et al., J. Exp. Med., 137: 85 (1973); Sullivan, L. M., et al., Cell, 45: 905 (1986); and Fenner, F. et al. in The Orthopoxviruses Academic Press (San Diego) (1989)).
In addition to preserving coagulation and fibrinolytic mechanisms (Furie, et al., Cell 53: 505-518 (1988)), certain proteases influence pleiotropic cellular responses, such as motility (Ossowski, Cell 52: 321-328 (1988)), differentiation (Bories, et al., Cell 59: 959-968 (1989)), and mitogenesis (Glenn, et al., Nature 278: 711-714 (1979); Kirchheimer, et al., PNAS USA 86: 5424-5428 (1989)), through the signalling properties of specialized protease receptors. As a result of ligand-dependent local proteolysis (Vu, et al., Cell 64: 1057-1068 (1991)) or physical receptor occupancy (Appella, et al., J. Biol. Chem. 262: 4437-4440 (1987)), protease receptors initiate complex pathways of cell activation with release of intracellular second messengers (Vu, et al., Id. (1991); Paris, et al., J. Biol. Chem. 259: 10989-10994 (1984)), protein phosphorylation (Golden, et al., J. Cell Biol. 111: 3117-3127 (1990)), and transcription of early activation genes (Daniel, et al., J. Biol. Chem. 261: 9579-9582 (1986)).
It has also been reported that abnormal expression of growth factor receptors contributes to certain neoplasias (Ulrich, et al., Nature 309: 418-425 (1984); Sherr, et al., Cell 41: 665-676 (1985); Downward, et al., Nature 307: 521-527 (1984)), but whether it also participates in human leukemogenesis remains uncertain. (See, e.g., Sawyers, et al., Cell 64: 337-350 (1991); Heard, et al., Cell 51: 663-673 (1987); Meeker, et al., Blood 76: 285-289 (1990).)
Various immune-inflammatory reactions are also affected by protease activity. The binding of urokinase as well as of thrombin to their complementary cellular receptors produces a potent chemotactic reaction with local accumulation of neutrophils and monocytes in vivo (Bovie, M. D. P., et al., J. Immunol., 139: 169 (1987); Bar-Shavit, R., et al., Science, 220: 728 (1983)). Moreover, synthetic protease inhibitors have been shown to decrease or abolish NK- and CTL-mediated target cell lysis, as well as monocyte synthesis and release of TNF-.alpha. (Redelman, D., et al., J. Immunol., 124: 870 (1980); Chang, T. W., et al., J. Immunol., 124: 1028 (1980); Suffys, P., et al., Eur. J. Biochem., 178: 257 (1988); Scuderi, P., J. Immunol., 143: 168 (1989)).
This concept of a more direct participation of proteases in specific cellular immune effector functions has recently been reinforced by the identification of a family of related serine proteases in cytotoxic NK and CTL clones (Masson, D., et al., Cell, 49: 679 (1987)). These serine proteases, termed granzymes (Jenne, D., et al., Proc. Natl. Acad. Sci. USA, 85: 4814 (1988)), are compartmentalized in subcellular granules together with the pore-forming protein perforin and are locally released during the polarized exocytosis associated with the formation of endothelial:T cell conjugates (Masson, D., et al., J. Biol. Chem., 260: 9069 (1985); Pasternack, M. S., et al., Nature, 322: 740.12 (1986); Podack, E. R., et al., J. Exp. Med., 160: 695 (1984)).
As revealed by molecular cloning, several granzymes share a remarkable degree of homology with other serine proteases involved in coagulation and fibrinolysis, and particularly with the plasma coagulation proteases factors IXa and Xa (Jenne, D., et al., Proc. Natl. Acad. Sci. USA, 85: 4814 (1988); Gershenfeld, H. K., et al., Science, 232: 854 (1986); Jenne, D., et al., J. Immunol., 140: 318 (1988); Lobe, C. G., et al., Science, 232: 858 (1986); Gershenfeld, H. K., et al., Proc. Natl. Acad. Sci. USA, 85: 1184 (1988)). While compelling evidence has accumulated suggesting a direct role for perforin in target cell injury (Masson, D., et al., J. Biol. Chem., 260: 9069 (1985), Duke, R. C., et al., J. Exp. Med., 170: 1451 (1989)), the participation and mechanistic role of the granzymes or other serine proteases in the lytic process remains unclear (Dennert, G., et al., Proc. Natl. Acad. Sci. USA, 84: 5004 (1987)).
It is also important to appreciate that the assembly of proteolytic activities on cellular surfaces initiates a variety of essential biologic responses. Specific high affinity receptors coordinate such interactions, protect the protease from inactivation by ubiquitous extracellular inhibitors, and provide optimal spatial alignment for the catalytic efficiency of the enzyme. The regulated association of coagulation and fibrinolytic proteins with a variety of cells may well exemplify these mechanisms of specialized protease-cell interactions (Miles, L. A., et al., Fibrinolysis, 2: 61 (1988); Morrissey, et al., Cell, 50: 129 (1987); Nesheim, M. E., et al., J. Biol. Chem., 254: 10952 (1979)).
Binding of the coagulation protease factor Xa to vascular cells was originally recognized as the molecular prerequisite for the assembly of the prothrombinase complex with membrane-bound factor V/Va (Tracy, et al., J. Biol. Chem. 260: 2119-2124 (1985); Rodgers, et al., PNAS USA 80: 7001-7005 (1983)). However, more recent studies have also postulated the existence of additional cell surface receptors for factor Xa, distinct from factor V/Va. Activated rabbit alveolar macrophages express a factor Xa receptor only in part immunologically related to factor V/Va, that promotes prothrombin activation in the absence of factor V/Va (McGee, et al., J. Exp. Med. 164: 1902-1914 (1986)).
Using monoclonal antibody (mAb) strategy, similar findings were independently reported on human monocytes and monocytic cells (Altieri, et al., J. Biol. Chem. 264: 2969-2972 (1989)). Membrane expression of this leukocyte factor Xa receptor, denominated Effector cell Protease Receptor-1 (EPR-1), was dynamically regulated by cell activation, with an 8- to 10-fold increased surface expression during lymphocyte proliferation in vitro (Altieri, et al., J. Immunol. 145: 246-253 (1990)). Although of lower affinity as compared with the factor Va:factor Xa interaction (Tracy, et al., Id. (1985)), binding of factor Xa to EPR-1 promoted prothrombin activation (Altieri et al., Id. (1989)) or the generation of intermediate products of factor IX activation (Worfolk, et al., Blood 80: 1989-1997 (1992)) at the monocyte surface.
The primary structure of EPR-1 has now been elucidated by functional cloning and mammalian cell expression in the cDNA. The results indicate that EPR-1 is a novel transmembrane glycoprotein receptor for factor Xa, potentially implicated in protease-dependent mechanisms of intracellular signal transduction. Anti-EPR-1 antibodies described herein have also been found to possess novel utilities and capabilities.